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β-cells regeneration by WL15 of cysteine and glycine-rich protein 2 which reduces alloxan induced β-cell dysfunction and oxidative stress through phosphoenolpyruvate carboxykinase and insulin pathway in zebrafish in-vivo larval model

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Abstract

Background

Pancreatic β-cells are susceptible to oxidative stress, leading to β-cell death and dysfunction due to enhanced ROS levels and type 2 diabetes. To inhibit the β-cells damages induced by the oxidative stress, the present study investigates the beneficial effect of various peptides (WL15, RF13, RW20, IW13 and MF18) of immune related proteins (cysteine and glycine-rich protein 2, histone acetyltransferase, vacuolar protein sorting associated protein 26B, serine threonine-protein kinase and CxxC zinc finger protein, respectively). Also, the molecular mechanism of WL15 from cysteine and glycine-rich protein 2 on β-cell regeneration was identified through PEPCK and insulin pathway.

Materials and methods

In this study, a total of five peptides including WL15, RF13, RW20, IW13, and MF18 were derived from immune-related proteins such as cysteine and glycine-rich protein 2, histone acetyltransferase, vacuolar protein sorting associated protein 26B, serine threonine-protein kinase and CxxC zinc finger protein, respectively. These protein sequences were obtained from an earlier constructed transcriptome database of a teleost Channa striatus. The identified peptides were evaluated for their antioxidant as well as antidiabetic activity. Based on the in silico analysis and in-vitro screening experiments, WL15 was predicted to have better antioxidant and antidiabetic activity among the five different peptides. Therefore, WL15 alone was further analyzed for apoptosis, antioxidant capacity, glucose metabolism, and gene expression performance, which was investigated on the alloxan (500 µM) induced zebrafish in vivo larval model.

Results

The results showed alloxan exposure to zebrafish larvae for a day, the ROS was generated in the β-cells. Interestingly, WL15 treatment showed a protective effect by reducing the toxicity of alloxan exposed zebrafish larvae by increasing their survival and heart rate. Moreover, WL15 reduced the intracellular ROS level and apoptosis in alloxan-induced larvae. The superoxide anion and lipid peroxidation levels are also reduced by improving the glutathione content after the WL15 treatment. Besides, WL15 treatment increased the proliferation rate of β-cells and decreased the glucose level. Further, the gene expression studies revealed that WL15 treatment normalized the PEPCK expression while upregulating the insulin expression in alloxan exposed larvae.

Conclusion

Overall, the findings indicate that WL15 of cysteine and glycine-rich protein 2 can act as a potential antioxidant for type 2 diabetes patients in respect of improving β-cell regeneration.

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Data availability

The data used to support the findings of this study are available from the corresponding author upon request.

Code availability

Not applicable.

Change history

Abbreviations

AGEs:

Advanced glycation end products

PEPCK:

Phosphoenolpyruvate carboxykinase

DHE:

Dihydroethidium

NDA:

NDA, naphthalene-2,3-dicarboxal-dehyde

T2D:

Type 2 diabetes

ROS:

Reactive oxygen species

DCFDA:

2'-7'-Dichlorofluorescein diacetate

DPPP:

Diphenyl-1-pyrenylphosphine

SD:

Standard deviation

EDTA:

Ethylenediaminetetraacetic acid

2-NBDG:

2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino]-2-deoxy-D-glucose

DCFDA:

2',7'-Dichlorodihydrofluorescein diacetate

CSRP2:

Cysteine and glycine-rich protein 2

HATs:

Histone acetyltransferase

VPS26B:

Vacuolar protein sorting associated protein 26B

STPK:

Serine threonine-protein kinase

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Acknowledgements

The authors extend their sincere appreciation to the Researchers Supporting Project Number (RSP-2021/191), King Saud University, Riyadh, Saudi Arabia.

Funding

Researchers Supporting Project Number (RSP-2021/191), King Saud University, Riyadh, Saudi Arabia.

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Authors and Affiliations

  1. Department of Biotechnology, College of Science and Humanities, SRM Institute of Science and Technology, Kattankulathur 603 203, Chennai, Tamil Nadu, India

    Ajay Guru, Gokul Sudhakaran & Jesu Arockiaraj

  2. Department of Zoology, College of Science, King Saud University, P.O. Box: 2455, Riyadh, 11451, Saudi Arabia

    Mikhlid H. Almutairi & Bader O. Almutairi

  3. Foundation for Aquaculture Innovations and Technology Transfer (FAITT), Thoraipakkam, Chennai, 600 097, Tamil Nadu, India

    Annie Juliet

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Contributions

AG and JA contributed to the concept and design of the study; AG and GS performed the experiments; MHA. BOA. AJ and JA contributed significantly to resources, data analysis, manuscript preparation and perform the analysis with constructive discussions; JA supervised and checked the manuscript; All authors read and approved the final manuscript.

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Correspondence to Jesu Arockiaraj.

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All authors declare that they have no conflict of interest.

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This research does not involve any human objects; however, we have performed few assays using zebrafish embryo and larvae. The fish were handled and experimented carefully as per the Institute Animal Handling Procedure and Ethical Approval and Clearence (No. SAF/IAEC/211215/004).

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The original online version of this article was revised: The affiliation of the author "Annie Juliet" is corrected as "Foundation for Aquaculture Innovations and Technology Transfer (FAITT), Thoraipakkam, Chennai 600 097, Tamil Nadu, India.

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Guru, A., Sudhakaran, G., Almutairi, M.H. et al. β-cells regeneration by WL15 of cysteine and glycine-rich protein 2 which reduces alloxan induced β-cell dysfunction and oxidative stress through phosphoenolpyruvate carboxykinase and insulin pathway in zebrafish in-vivo larval model. Mol Biol Rep 49, 11867–11879 (2022). https://doi.org/10.1007/s11033-022-07882-4

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